This application requests funds to purchase a dedicated AB Sciex Qtrap 6500 LC-MS/MS system to allow high-sensitivity analysis of lipid metabolites in complex samples, as well as to enable rapid, automated analysis of samples generated by the Vanderbilt High Throughput Screening Facility (VHTS). The instrument will be housed within and operated by the Mass Spectrometry Core of the Vanderbilt Mass Spectrometry Research Center (MSRC). It will support the research of a group of major users, including Lawrence Marnett, Sachin Patel, and Roger Colbran, who study the chemistry and biology of endocannabinoid metabolism in response to pain, inflammation, and stress. Their work requires quantification of the endocannabinoids, 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (AEA), and their metabolites formed by hydrolysis or oxygenation. Major user Ned Porter studies the chemistry and biochemistry of 7-dehydrocholesterol, an intermediate in cholesterol biosynthesis that is highly susceptible to oxidation. His work requires quantification of this sterol and its numerous oxidation products. All of these investigators employ in vivo models that provide limited amounts of material for quantification of low abundance analytes. The instruments currently available at Vanderbilt are not sensitive enough to enable detection of many of the analytes of interest. Furthermore, the major users require rapid data-dependent scanning that can employ a variety of tandem MS experiments as the survey scan for planned metabolomics experiments. The fifth major user of the QTrap 6500 will be the VHTS, which has incorporated early-stage metabolic assays to assess the potential in vivo stability of novel bioactive compounds. These assays are done in high-throughput format (96-well and 384-well plates), which provides large numbers of samples for analysis that contain limited amounts of metabolites. These studies are critical to the medicinal chemistry efforts of a growing number of NIH-funded investigators, who need them to maximize the in vivo stability of bioactive compounds for use as chemical probes of mechanism and therapeutic proofs-of-concept. The limited access of the VHTS group to high sensitivity mass spectrometry has precluded routine adoption of the metabolic assays so critical for compound translation. The QTrap 6500 LC-MS/MS will be subject to the operating guidelines of the MSRC, which will also take responsibility for its maintenance and user training. The Major user group will account for approximately 76% of the use of the instrument, with the remainder made available to other Vanderbilt investigators requiring high sensitivity analysis. The Mass Spectrometry Core Advisory Committee will monitor instrument use and establish criteria for access by non-major users.